Method and apparatus for generation of microparticles containing immobilized enzyme

ABSTRACT

The invention concerns a method and an apparatus for generation of micropartides containing an immobilized functional component, where the following measures are proposed: —spraying a liquid ( 32 ) containing a soluble alginate and a functional component consisting of molecules or nanoparticles to generate a stream ( 60 ) of droplets, —directing the stream ( 60 ) of droplets onto a precipitation bath ( 16 ) and capturing the droplets therein by application of high voltage ( 14 ), —precipitating the droplets in the precipitation bath ( 16 ) by means of a precipitation liquid ( 18 ) containing an alginate complexing agent, such that the droplets are solidified to form micropartides ( 10 ) containing the functional component and —extracting the micropartides ( 10 ) from the precipitation bath ( 16 ).

The invention concerns a method and an apparatus for generation ofmicroparticles containing an immobilized functional component,specifically an enzyme or other functional molecules or sub-micro-sizedparticles (nanoparticles). The invention further concerns a diagnostictest element, specifically glucose test element.

In diagnostic test elements or sensors for glucose tests it is known toprovide enzymes in a reagent layer or electrode layer to induce chemicalreactions which are responsive to an analyte in a body fluid contactedwith the test element. Such analyses are usually made with handhelddevices on the spot by patients themselves. There, it should beguaranteed that the enzymes are immobilized without skin contact, andthat the enzymes can be reached by diffusion of the sample ingredientsin a given measurement time. In this context, it is known to include theenzymes in a wet chemistry composition which is applied on a substrateand further coated after drying.

On this basis the object of the invention is to further improve theknown methods and for generation of microparticles and to provideimproved diagnostic test elements specifically for self-testing glucosemeasurement systems.

The combination of features stated in the independent claims is proposedto achieve this object. Advantageous embodiments and furtherdevelopments of the invention are derived from the dependent claims.

The invention is based on the idea of generating solidifiedmicroparticles as a carrier for at least one immobilized functionalcomponent, which has a specific function in a diagnostic test.Accordingly it is proposed according to the invention that a method forgeneration of microparticles comprises the steps of

-   a) spraying a liquid which contains a soluble alginate and a    functional component consisting of molecules or nanoparticles to    generate a stream of droplets, wherein spraying the liquid comprises    applying a gas stream to the liquid, thereby atomizing the liquid in    the gas stream,-   b) directing the stream of droplets onto a precipitation bath and    capturing the droplets therein by application of high voltage,-   c) precipitating the droplets in the precipitation bath by means of    a precipitation liquid containing an alginate complexing agent,    whereby the droplets are solidified to form microparticles    containing the functional component, and-   d) extracting the microparticles from the precipitation bath.

In such a way, it is possible to create particles from a physiologicalnon-hazardous material and of very small size, i.e. at most 100 microns,so that there is provided a comparatively large surface for the analytesto reach the functional component which is immobilized in the particlesstructure. An adequate particle size can be achieved by atomizing aliquid and by directing a mist-like stream of droplets under theinfluence of a high voltage to a precipitation bath, such that aconsiderable yield is achieved and the spray is not lost before reachingthe precipitation liquid. By atomizing the liquid in the gas stream,there is no need for a complex electrostatic atomization technique.Moreover, such a gas stream allows adjustment of the droplet size bysimple measures such as adjusting the velocity and temperature of thecarrier gas. Thus, the generation of a stream of droplets in a gasstream provides further degrees of freedom: The droplet size can beadjusted independent of the voltage of a potential subsequentelectrostatic charge. By means of increasing the gas pressure, it ispossible to decrease the average diameter of the droplets, even below adimension which is achievable by electrostatic atomization of a givenliquid, e.g. to 2-10 μm. The droplet dimension is not influenced,neither by the distance to the precipitation bath nor by the electricconductivity of the initial solution nor by the magnitude of an appliedhigh voltage. Furthermore, it is possible to achieve a high throughputwith simple measures.

According to a preferred embodiment, the molecules forming thefunctional component are selected from the group of enzymes, coenzymes,mediators, stabilizers and dyes, and preferably are enzymes.

It is also preferred that the nanoparticles forming the functionalcomponent are sized in at least one dimension, preferably in 3dimensions, below 1 μm. The nanoparticles may preferably be selectedfrom the group of metal, metal alloy, metal oxide and carbon.

Advantageously also with respect to a simplified arrangement, sprayingthe liquid in the afore-mentioned step a) comprises applying a gasstream to the liquid provided in a reservoir.

For further improvement of the yield of generated particles, it isadvantageous when a moving surface is provided in the precipitationbath, wherein the moving surface is continuously loaded with a film ofthe precipitation liquid and the droplets are disposed onto the movingsurface such that agglutination of droplets or particles is avoided.

Another improvement provides that a target electrode connected to thehigh voltage is arranged in the precipitation bath, specifically in theform of a submerged rotating drum. This allows to provide a focusedattractive force in addition to gravitation.

For further production improvement it is advantageous when anelectrically conductive nozzle is connected as an electrode to the highvoltage, and the stream of previously generated droplets is directedthrough the nozzle.

Surprisingly, the electrostatic charging of the droplets within the gasstream during passage of the high voltage nozzle works without problemsand efficiently. Without the need for a direct contact with the nozzle,an electrostatic charging of the droplets occurs, which is used to bringthe droplets in effective contact with the precipitation bath. Due tothe electric charge, an attractive force occurs in direction to theinversely poled precipitation bath, which brings the micro-droplets incontact with the bath surface.

In a particular embodiment the microparticles are formed with adimension of less than 50 μm, e.g. in the range from 1 to 20 μm,preferably less than 20 μm, e.g. in the range from 1 to 10 μm, and morepreferably less than 10 μm, e.g. in the range from 0.1 to 5 μm.Microparticles of such a small size provide sufficient surface as adiffusion interface, and with a limited size distribution preferably inthe single-digit micron range it is possible to achieve a homogenousresponse.

Advantageously, the method further comprises the step of separatingmicroparticles of different size by an ion exchange process usingdifferent ions in the alginate complexing agent, specifically bariumions and calcium ions.

For further improvement of the practical value it is advantageous whenthe microparticles are provided with a stabilizing shell made of apolymer material, specifically of polycations.

An intermediate product can be generated for further processing bydischarging a suspension of the microparticles through an outlet of theprecipitation bath.

It is also favorable when the microparticles are treated in a cleaningbath by means of an ion exchange process, specifically to removebiological hazardous substances.

In regard to a preferred use it is advantageous when the microparticlesare deposited in a layer of a diagnostic test element, specifically in areagent layer or electrode layer of a glucose test element.

With regard to an apparatus adapted for generation of microparticlescontaining an immobilized functional component, in order to solve theaforementioned object, it is proposed to provide a modular arrangementcomprising the following components:

-   -   a spraying unit adapted to generate a stream of droplets of a        liquid containing a soluble alginate and the functional        component, wherein the stream of droplets is generated by        applying a gas stream to the liquid, thereby atomizing the        liquid in the gas stream,    -   a high voltage unit adapted for charging and directing the        droplets to a target electrode and    -   a precipitation bath containing the target electrode and an        alginate complexing agent adapted to precipitate the droplets,        such that the droplets are solidified to form microparticles,    -   means adapted to extract the microparticles from the        precipitation bath.

A further improvement comprises an electrically conductive nozzle whichis connected as an electrode to the high voltage unit, wherein thestream of previously generated droplets is directed through the nozzlein order to charge the droplets.

Another aspect of the invention concerns a diagnostic test element,specifically glucose test element, comprising a reagent layer orelectrode layer, wherein said layer comprises microparticles containingat least one immobilized functional component which consists ofmolecules or nanoparticles.

The proposed inclusion of microparticles is particularly effective whena two-layer structure comprising an intermediate and an outer layer isapplied on a carrier substrate, and when the microparticles are includedin the intermediate layer.

Advantageously, the molecules forming the functional component areselected from the group of enzymes, coenzymes, mediators, stabilizers,and dyes.

Further advantageously and specifically tailor-made for blood glucosetests, the at least one functional component is an enzyme which isselected from the following group of enzymes: oxi-reductase-enzymes,e.g. GlucDOR/PQQ (Glucose Dye Oxidoreductase), dehydrogenase-enzymes,e.g. glucose dehydrogenase, in particular NAD-dependent glucosedehydrogenase, FAD-dependent glucose dehydrogenase and/or PQQ-dependentglucose dehydrogenase, oxidase-enzymes, e.g. glucose oxidase.

Generally, the sub-micro-sized particles or nanoparticles forming thefunctional component are considerably smaller than the microparticlesand have a diameter of less than 1 μm, preferably 1 to 500 nm,especially preferred of 1 to 100 nm.

The material of the nanoparticles usually is one of metal and metalalloy and metal oxide and carbon.

When acting as a catalyst, the nanoparticles are selected of at leastone of platinum and palladium and silver and rhenium and rhodium andiron and nickel and cobalt and copper and chromium and zinc andaluminium and manganese and molybdenum or alloys thereof or oxidesthereof.

Alternatively, the nanoparticles are quantum dots acting as afluorescent dye.

As a further alternative, the nanoparticles are an electric conductorselected out of metals, metal oxides or carbon, e.g. in the form ofnanowires or nanotubes acting as a contributor to the electricconductivity of the reagent layer.

The invention is further elucidated in the following on the basis of anembodiment example shown schematically in the drawings, where

FIG. 1 is a schematic view of an apparatus for generation ofmicroparticles;

FIG. 2 is sectional view of a layered diagnostic test element comprisingmicroparticles.

FIG. 1 illustrates a system or apparatus for generation ofmicroparticles 10 comprising a spraying unit 12, a high voltage unit 14,a precipitation bath 16 including a precipitation liquid 18 and arotatable drum 20, and extraction means 22 for extracting precipitatedmicroparticles 10 from the bath 16.

The spraying unit 12, which can be positioned over the precipitationbath 16 by means of a stand 24, is provided with a gas inlet 26, anatomizer chamber 28 including a reservoir 30 for a feed liquid 32 and anaerosol outlet 34 ending in a discharge nozzle 36. The feed liquid 32contains an aqueous solution of a soluble alginate, e.g. sodium alginateand at least one of enzyme selected from oxi-reductase-enzymes,dehydrogenase-enzymes or oxidase-enzymes and other functional molecules.Therewith, it is finally aimed to form beads of alginate complexescontaining immobilized enzymes as (spherical) microparticles with a sizeor dimension (diameter) in the range from 1 to 20 μm.

The high voltage unit 14 has a source 38 for a d.c. voltage in the rangeof 3 to 80 kV, preferred 10 to 60 kV which is supplied between a firstport 38 and a second port 40. First port 38, suitably grounded, isconnected to the drum 20 which thereby forms a target electrode 42,whereas second port 40 is connected to the nozzle 36 forming a counterelectrode 44. Optionally, the first port 38 may be connected to theprecipitation liquid 18, and second port 40 may be connected to chargethe feed liquid 32.

The precipitation bath 16 comprises an open-top container 46 which hasan inclined bottom plane 48 for guiding precipitated microparticles 10to a discharge connection 50 of the extraction means 22.

The horizontally oriented drum 20 is rotatable around its center axis bymeans of a motor 52, where the output shaft 54 is arranged along thefluid level of the precipitation liquid 18. In this way, the cylindricalmantle of the rotated drum 20 is partially submersed and hence forms amoving surface 56 which is continuously loaded with a revolving film ofthe precipitation liquid 18.

The system may comprise a central control unit (not shown) forcontrolling the operating procedure and the process parameters of thevarious system units.

In use, the inlet 26 of the spraying unit 12 is loaded with a stream 58of a carrier gas, e.g. air provided from a compressor. Under the effectof the gas stream 58, the feed liquid 32 is atomized into nebulardroplets. Advantageously, the size of the droplets is adjusted by thevelocity, temperature and humidity of the carrier gas, whereinevaporation leads to a miniaturization of the droplets during flight. Inthis stage, a fine aerosol jet 60 is generated by non-electric effects.

Then, the jet 60 is ejected through the nozzle 36, where the dropletsare electrically charged to a high potential. The nozzle 36 directs thejet 60 against the drum 20, where the charged droplets are attracted bythe target electrode 42 and captured in the film of the precipitationliquid 18 coating the mantle or moving surface 56.

The precipitation liquid 18 contains an aqueous solution of an alginatecomplexing agent including e.g. Ba²⁺ ions which permeate the dropletsand lead to solidified beads or microparticles 10 containing immobilizedenzyme(s).

In addition to the complexing agent, further ingredients may be providedin the precipitation liquid 18 to structurally stabilize the developingbeads on their surface. This may be achieved by polymers, specificallypolycations, which preferentially adsorb on the surface of the beads andform a complex with the solidifying alginate component thereby providinga stabilizing outer shell.

The solidified beads which sediment on the inclined bottom plane 48 areguided to the discharge connection 50, where a suspension ofmicroparticles 10 can be discharged to the extraction means 22 either ina continuous or a batch mode.

It is also conceivable to separate microparticles of different size by acation ion exchange process. When the microparticles 10, which wereprecipitated with ions of comparatively high atomic mass, are fed into asolution of a precipitating ion of lower atomic mass, e.g. Ca²⁺ in asaturated CaSO₄-solution, an ion exchange process occurs with theresulting beads having a lower density. As this happens faster onsmaller particles having a comparatively larger surface, smaller beadswill ascend in the solution, and a separation can be achieved bydecantation. At the same time, the ion exchange process leads to acleaning in the sense of a reduced toxicity of Ca²⁺-containingmicroparticles 10.

The microparticles 10 containing immobilized enzyme(s) are particularlyuseful in diagnostic test elements designed for glucose tests. Such testelements may be provided on disposable test tapes or test strips, eitherfor optical or electrochemical analyses.

FIG. 2 shows a sectional view of a glucose test element 62 which isgenerally designed as a two-layered composite on a substrate 62 formedby a transparent plastic carrier. The substrate 62 is coated with afirst layer 66 of a reactive test material. This material containsmicroparticles 10 including immobilized enzymes and some of functionalmolecules as mediator and dye. The latter reacts by a color changeinduced by the enzymes which are responsive to glucose. In one exemplarycomposition, oxidase- or dehydrogenase enzymes are used, e.g. glucoseoxidase or glucose dehydrogenase, and the dye includes molybdenum in theform of phosphomolybdic acid.

The first layer 66 is covered by a second layer 68 of test materialcontaining most of the mediator and of the dye which are also present inthe first layer. Further, the second layer 68 contains white pigmentsfor separation of a blood sample and for providing a white backgroundfor optical measurement of the color change. It is notably important toavoid that enzymes and other functional molecules permeate the opticalbarrier formed by the second layer 68 specifically during drying of therespective wet chemistry composition. The microparticles 10 immobilizethe enzymes in such a way that they cannot reach the second layer 68during the manufacturing or analysis process.

On the upper side of the test element 62, a spreading web 70 is attachedfor homogenous and planar distribution of a blood sample. Whenconducting a diagnostic test, the blood sample is applied by the user asa droplet from a skin wound.

1. A method for generation of microparticles containing an immobilizedfunctional component, the method comprising the steps of a) spraying aliquid which contains a soluble alginate and a functional componentconsisting of molecules or nanoparticles to generate a stream ofdroplets, wherein spraying the liquid comprises applying a gas stream tothe liquid, thereby atomizing the liquid in the gas stream, b) directingthe stream of droplets onto a precipitation bath and capturing thedroplets therein by application of high voltage, c) precipitating thedroplets in the precipitation bath by means of a precipitation liquidcontaining an alginate complexing agent, such that the droplets aresolidified to form microparticles containing the functional component,and d) extracting the microparticles from the precipitation bath.
 2. Themethod of claim 1, wherein the molecules forming the functionalcomponent are selected from the group of enzymes, coenzymes, mediators,stabilizers and dyes, and preferably are enzymes, and/or wherein thenanoparticles forming the functional component are sized in at least onedimension below 1 μm and/or are selected from the group of metal, metalalloy, metal oxide and carbon.
 3. The method of claim 1, whereinspraying the liquid in step a) comprises applying the gas stream to theliquid in a reservoir.
 4. The method according to claim 1, furthercomprising providing a moving surface in the precipitation bath, whereinthe moving surface is continuously loaded with a film of theprecipitation liquid and the droplets are disposed onto the movingsurface.
 5. The method according to claim 1, wherein a target electrodeconnected to the high voltage is arranged in the precipitation bath,specifically in the form of a submerged rotating drum.
 6. The methodaccording to claim 1, wherein a nozzle is connected as an electrode tothe high voltage, and the stream of droplets is directed through thenozzle.
 7. The method according to claim 1, wherein the microparticlesare formed with a dimension of less than 50 μm, preferably in the rangefrom 1 to 20 μm.
 8. The method according to claim 1, further comprisingseparating microparticles of different size by an ion exchange processusing different ions in the alginate complexing agent, specificallybarium ions and calcium ions.
 9. The method according to claim 1,further comprising providing the microparticles with a stabilizing shellmade of a polymer material.
 10. The method according to claim 1, furthercomprising discharging a suspension of the microparticles through anoutlet of the precipitation bath.
 11. The method according to claim 1,further comprising cleaning of the microparticles in a cleaning bath bymeans of an ion exchange process.
 12. The method according to claim 1,further comprising depositing the microparticles in a layer of adiagnostic test element, specifically in a reagent layer or electrodelayer of a glucose test element.
 13. An apparatus for generation ofmicroparticles containing an immobilized functional component, whereinthe functional component consists of molecules or nanoparticles,comprising a) a spraying unit adapted to generate a stream of dropletsof a liquid containing a soluble alginate and the functional component,wherein the stream of droplets is generated by applying a gas stream tothe liquid, thereby atomizing the liquid in the gas stream, b) a highvoltage unit for directing the droplets to a target electrode, c) aprecipitation bath containing the target electrode and an alginatecomplexing agent adapted to precipitate the droplets, such that thedroplets are solidified to form microparticles, and d) means adapted toextract the microparticles from the precipitation bath.
 14. Theapparatus of claim 13, further comprising an electrically conductivenozzle which is connected as an electrode to the high voltage unit,wherein the stream of previously generated droplets is directed throughthe nozzle.
 15. Diagnostic test element, specifically glucose testelement, comprising a reagent layer or electrode layer, wherein saidlayer comprises microparticles containing at least one immobilizedfunctional component which consists of molecules or nanoparticles,wherein the microparticles are preferably generated by a methodaccording to claim
 1. 16. The diagnostic test element of claim 15,wherein a two-layer structure comprising an intermediate and an outerlayer is applied on a carrier substrate, and the microparticles areincluded in the intermediate layer.